1. Field of the Invention
The invention relates generally to assays for drugs in blood and other biological fluids. More particularly, it relates to isolated binding proteins for the immunosuppressive drug, rapamycin, and the use of these proteins in a quantitative competitive protein binding assay for rapamycin in blood and other biological fluids.
2. Description of the Background Art
Rapamycin ("RAP") is an antifungal macrocyclictriene antibiotic extracted from Streptomyces hygroscopicus isolated from an Easter Island soil sample.sup.1. It is highly active against Candida species, especially Candida albicans. See, formula (1) below.
Early animal studies demonstrated that RAP is immunosuppressive; it totally prevented the development of experimental immunopathies and the formation of IgE-like antibody in rats.sup.2.
Since these early reports, RAP has received little attention. However, interest in this molecule has recently been rekindled with the discovery that the macrolide antibiotic, FK-506, a product of Streptomyces tsukubaensis.sup.3, bears structural similarity to RAP.sup.4,5 (1). ##STR1##
FK-506 is capable of inhibiting immune responses with a potency some 100-500 fold greater than that of cyclosporine A.sup.6 ("CsA"), currently the favored therapeutic agent for prevention of graft rejection after organ and bone marrow transplantation.sup.7-9. FK-506 has performed remarkably well in initial human clinical transplantation trials.sup.10,11, despite reports of toxic effects in animals.sup.12.
RAP inhibits T cell activation at concentrations comparable to those of the structurally related FK-506, yet with mechanisms reportedly strikingly different from those mediated by FK-506, and thus cyclosporine A.sup.13. A bright future for RAP as a clinically useful immunosuppressive agent is predicted.
The reportedly predominant cyclosporine binding protein is a cytosolic molecule of Mr 17,739 termed cyclophilin.sup.14. However, high specific binding of CsA to cytosolic proteins larger (e.g., 50-58 kDa) and smaller (e.g., 10-12 kDa) than cyclophilin have been identified in circulating and cultured lymphocytes, isolated, and used as a reagent in a competitive protein binding assay for CsA and its biologically active metabolites.sup.15-17.
Binding of CsA to a 50 kDa protein was found to correlate well with a bioassay.sup.17,18.
The reportedly predominant FK-506 binding protein in calf thymus, human spleen and the T cell line JURKAT, termed FKBP, is a heat stable protein of mass of about 10 kDa to 12 kDa.sup.19-23. However, I have identified and isolated specific receptor proteins for FK-506 in the range of 40 kDa to 60 kDa, particularly 51 kDa, and made the 51 kDa protein the basis of a competitive protein binding assay for FK-506 in biological fluids.sup.24,25.
Both cyclophilin and FKBP are reported to exhibit peptidyl-prolyl isomerase activity.sup.21,26. This enzyme activity is inhibited by CsA and FK-506, respectively.
Recently, FKBP isolated from yeast, calf thymus and human T cells (JURKAT) has been shown to bind RAP with a Kd of about 0.2 nM, greater than the Kd (0.4 nM) for FK-506, and RAP is also reportedly a potent inhibitor of the aforementioned isomerase activity of FKBP.sup.27.
There currently is no quantitative assay for RAP in blood and biological fluids suitable for use in a clinical setting. The only reported assay for RAP in serum or tissues is a microbiological bioassay.sup.28. Such assays are far too cumbersome, time consuming and expensive, and with far too little throughput of data, to be useful in a hospital clinical laboratory setting. The development of a suitable assay is critical to the clinical use of RAP.
The critical need in medicine for a rapid and specific assay for rapamycin and its biologically active metabolites, derivatives and analogues in blood and other biological samples is now filled by the competitive protein binding assay disclosed and claimed below.